Fluorescent Peptomer Substrates for Differential Degradation by Metalloproteases

Abstract

Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide−peptoid hybrids) to probe the impact of nonnatural residues on MMP specificity for an MMP peptide consensus sequence. Peptoids are non-natural, N-substituted glycines with a large side-chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offer N-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography−mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well tolerated in the P3 and P3′ substrate sites and that the identity of the peptoid in these sites displays a moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with side-chain identity than sidechain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as "fingerprints" for the proteases and yielded separation by multivariate data analysis. To further demonstrate the practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.

Microbial proteases and their applications

“…More sensitive methods are needed when the quantity or activity of protease enzymes are low, and sensitive fluorescent peptide substrates are available, through which the limit of detection reaches the ng level ( Austin et al, 2022 ).…”

“…However, when detection is performed using a single fluorescently labeled protease substrate, the product and substrate must be separated, and the pH needs to be adjusted to enhance the detection signal. The detection steps remain relatively complex ( Twining, 1984 ; Austin et al, 2022 ).…”

Systematic d-Amino Acid Substitutions to Control Peptide and Hydrogel Degradation in Cellular Microenvironments

“…Several generalizable strategies have been proposed to control the degradation rate of the peptides, such as modifying the peptide sequence by introducing a d -amino acid (D-AA), − scrambling the sequence, switching amino acids, and introducing non-natural amino acids or peptoids in the sequence. Other strategies include modifying the structure of the peptide sequence by alkylation, cyclization, and glycosylation of the amino acids to impart proteolytic resistance.…”

Peptomer substrates for quantitative pattern-recognition sensing of proteases

“…We have previously demonstrated the utility of peptomers (peptide–peptoid hybrids) for differentiating between matrix metalloproteinases; 26 however, classification was only achieved at a single concentration condition. Here, our objectives were to (1) expand the breadth of proteases from different sources and families, (2) accurately classify protease identity over a range of concentrations, and (3) predict concentrations of proteases based on their degradative behavior.…”